【摘要】 目的克隆mic2/CD99基因并表达于L428细胞株。方法通过PCR及双酶切方法,从Jurkat细胞基因组RNA中获得mic2/CD99全长基因编码序列,克隆到pcDNA3.1(+)质粒载体上,构建包含mic2/CD99全长基因载体;用脂质体转染法将mic2/CD99全长基因载体转染至L428细胞系;并从分子水平验证其存在。结果RT-PCR方法扩增出大小为558bp片段,序列测定其编码序列正确,酶切鉴定亚克隆序列正确。结论成功构建了mic2/CD99全长基因载体,并稳定转染于L428细胞株中。更多还原

【Abstract】 Objective To construct a eukaryotic expression vector of CD99 gene for transfection into Hodgkin lymphoma L428 cells. Methods The full-length cDNA of CD99 gene was amplified from Jurkat cells by RT-PCR and cloned into the pcDNA3.1 (+) vector and transfected into L428 cell line using Lipofextamine 2000. The sequence of CD99 mRNA in the transfected cells was confirmed by restriction endonuclease digestion and DNA sequencing, and the expression of CD99 protein was identified using immunocytochemistry. Results A gene fragment of 558 bp was amplified from the transfected cells and the sequence was verified by DNA sequencing. Immunocytochemistry identified the presence of CD99 expression in the transfected cells. Conclusion A eukaryotic expression vector pcDNA3.1(+)-CD99 is successfully constructed and stably expressed in L428 cell line.更多还原